Over the last two decades there has been a growing interest in accelerating the processing steps of tissue biopsies that are embedded in paraffin for diagnostic evaluation. Clinical and research analysis of tissue samples is an ongoing science. The major forces driving reduced processing times are improved patient care and the control of rising medical costs. Equipment has been on the market for a number of years to reduce all the processing steps, to include slide preparation, of a surgical biopsy except for formalin fixation. It is well known in the art that formalin remains the most popular fixative for light microscopy and pathology. It is the fixative that defines the morphological basis for diagnostic pathology.
Tissue samples are routinely placed in plastic tissue processing cassettes, after grossing, for fixation in formalin. Grossing is the act of taking a large tissue sample and cutting it down into smaller tissue sizes.
It is well know in the art that fixation is the single most important step in processing of tissue for clinical and research pathology because:                (1) It stabilizes tissue proteins and other cellular constituents to withstand the rigors of processing; and        (2) Without adequate fixation the quality of results used for diagnostic evaluation are subject to compromise.        
Ironically, the least controlled step in tissue processing for samples derived from clinical or research pathology is fixation. As is well known in the art, processing—after fixation—is done by either automatic tissue processors or MW-assisted processing. Either process cycles the tissue cassettes through dehydration (usually a graded series of ethanol), an intermedium (usually xylene, isopropanol or xylene substitutes), and molten paraffin in a controlled environment.
The only piece of equipment, known to the inventors, that has been exploited to optimize and accelerate the fixation step is the MW oven. Formalin, used as a fixative, is typically diluted to a concentration of 10% and buffered to maintain a desired Ph. Formalin is a 37-40% solution of formaldehyde. 10% NBF (neutral buffered formalin) is a buffered 1:10 dilution of formalin concentrate (approximately 4% formaldehyde solution) and approximately 1% methanol. As a fixative, formalin is known for its ability to quickly penetrate tissue, but it is also known in the art that, once in the tissue, chemical stabilization (fixing and/or cross-linking proteins) of the tissue takes places slowly. Formalin fixation is generally considered, by those knowledgeable in the art, to take at least 24 hours or longer for tissue biopsies and requires 4-6 hours just to penetrate tissue blocks up to 5 mm thick.
As known in today's art, MW methods to optimize and/or accelerate the fixation process have been subdivided into 5 classifications:                1) MW stabilization—tissue placed in a non-fixative solution, usually normal saline, and heated to temperatures between 45 and 70° C. in the MW—it can also apply to tissues not placed in a solution and stabilized by MW's alone;        2) MW-assisted chemical fixation—tissue placed in a fixative solution and irradiated—a process, to the inventors knowledge, that is only used for tissues being processed for electron microscopy;        3) MW-assisted chemical fixation followed by immersion of tissue in fixative outside the MW;        4) Chemical fixation of the tissue outside the MW for minutes or hours followed by MW-assisted chemical fixation; and        5) MW irradiation used in combination with freeze fixation.        
MW stabilization, #1 above, produces rapid results (under 10 minutes) but suffers from a number of disadvantages unique to the process. Other than a substitute for the use of formalin there seems to be little interest in the process in the United States. MW-assisted chemical fixation, #2 above, has worked well with a number of aldehyde fixatives and fixative combinations for samples less than 1 mm3 in size and almost always destined for electron microscopic evaluation. Other than endoscopic or needle biopsies, which conform to the 1 mm3 size parameters, this method has not been described as a means of processing for clinical or research pathology. MW-assisted chemical fixation followed by immersion, #3 above, in the fixative outside the MW is a method used to circumvent the reported problems inherent with MW-assisted chemical fixation. Chemical fixation of the tissue for a period outside the MW, #4 above, is primarily a method used in clinical and research pathology to overcome the problems associated with the fixation of fresh tissue in formalin (see below). MW irradiation used in combination with freeze fixation, #5 above, is an established method for improving the morphology of cryostat sections.
MW-assisted formalin fixation of fresh tissue has been attempted without success. This lack of success is attributed to the mechanism of formalin fixation, as described in literature of the present art.
The following is the proposed mechanism steps of formalin fixation:                1) The diffusion of methylene glycol into the tissue—formaldehyde fixative solutions contain little formaldehyde, instead the primary chemical reagent is methylene glycol formed by the reaction of formaldehyde gas and water;        2) The formation of formaldehyde, the fixation component, by the dehydration reaction of methylene glycol in the tissue; and        3) Binding of formaldehyde to the proteins by chemical cross-linking of the formaldehyde to tissue proteins and other cellular components.        
When MW or other heating methods heat formalin, it is thought that steps 2 and 3, as outlined above, take place. So for fresh tissue, the heating (MW or other) results in the formation of formaldehyde wherever methylene glycol is present in or around the tissue. The resulting formaldehyde cross-links with the proteins in the tissue that creates a dense matrix, stopping further penetration of the formalin into the tissue. It has been demonstrated in a model system that aldehyde cross-linking takes place when the system is exposed to MW irradiation. It has also been demonstrated that dehydration reactions take place under the influence of MW heating. From the experience of the inventors, the penetration of formalin in fresh tissue is limited to approximately the outer 0.75 mm of the tissue when the MW is used at full power. During a workshop at the National Society for Histotechnology meeting (1998, Salt Lake City, Utah) the inventors demonstrated for the first time that variable power control and intermittent MW energy could be used to fix fresh tissue in formalin with MW irradiation.
The primary variable responsible for MW-accelerated fixation in the literature is attributed to MW induced heating of either a fixative or solution. The present invention provides an alternative that has not been previously described in the literature. This alternative of the present invention expands on previous knowledge by:                1) Using continuous MW energy initially at a low power output of the magnetron (typically at or below 250W);        2) Changing the continuous power output of the magnetron after approximately 75% of the total time has elapsed to a higher level of continuous power at or above 450W;        3) (1) and (2) are performed while circulating and cooling the formalin so that MW heating is held within in a narrow range (±0.5° C.);        4) (1) and (2) are performed while controlling formalin temperatures during MW irradiation between 4° C. and 40° C.; and        5) Proposing a plausible method by which this process takes place.        